Hematopoietic cell transplantation (HCT) from normal donors is a curative therapy for several inherited and acquired disorders. However, the transplant is limited by the poor availability of matched donors and the mortality associated with the allogenic procedure (mostly related to graft versus host disease—GvHD). HCT has a very low efficacy in some disorders such as lysosomal storage diseases (LSD). In order to improve the safety and efficacy of allogeneic transplants and to identify alternative protocols for patients lacking a matched donor, a gene therapy approach based on the transplantation of gene corrected, autologous hematopoietic stem cell (HSC) is required.
As an alternative to allogeneic HCT an inherited genetic defect can be corrected in the patient's own hematopoietic cells by gene therapy. However, delivery of a functional copy of the relevant gene into all affected cells of the body is difficult. The concept of stem cell gene therapy is based on the genetic modification of a relatively small number of stem cells, which remain long-term in the body by undergoing self-renewal divisions, and generate huge numbers of genetically corrected progeny, thus ensuring a continuous supply of corrected cells for the rest of the patient's lifetime. Hematopoietic stem cells (HSC) constitute an excellent target population for gene therapy, since they can be easily and safely obtained from bone marrow (BM) or mobilized peripheral blood. The isolated HSC can be genetically modified and returned to the patient as an autologous transplant. Long-term benefit requires the transplantation of a sufficiently high number of gene-modified HSC, which can repopulate the conditioned BM, giving rise to corrected blood cells of all hematopoietic lineages. Autologous allogeneic HSC make the transplant procedure available to all patients and avoids immunological compatibility problems leading to GvHD. In addition, some diseases like primary immunodefficiencies require the correction of a fraction of HSC and their progeny. The intensity of the conditioning regimen (so-called “non-myeloablative” or “mini” conditioning regimen) is reduced which results in better tolerability and fewer side effects for the patient. A reduced conditioning regimen is less compatible with a standard allogeneic transplant, because mixed donor chimerism is usually unstable in the allogeneic setting due to immunological antagonism with host-derived immune cells.
Efficient long-term gene modification of HSC and their progeny requires a technology which permits stable integration of the corrective DNA into the genome, without affecting HSC function. The most efficient delivery systems are viral vectors.
For example, gene transfer and expression in hematopoietic progenitor cell (HSPC) of the lysosomal enzyme galactocerebrosidase (lacking in Globoid Leukodystrophy—GLD— or Krabbe disease) causes apoptosis and functional impairment of the transduced cells, preventing the development of HSPC based gene therapy approaches for treating the disorder (see below). Thus, future expression cassettes used for gene therapy should resemble physiologic expression patterns and avoid ectopic and/or non-physiologic transgene expression, which can result in toxicity, elimination or even malignant transformation of the transduced cells. This is particularly important for stem cells, the key target cell type guaranteeing long-term efficacy of gene therapy, whose biology must not be disturbed by the genetic intervention.
To summarize, current hematopoietic gene therapy strategies require transduction of HSC to guarantee long-term correction of the hematopoietic system, but would significantly benefit from regulated transgene expression cassettes that do not ectopically express the transgene product in HSC, but “switch on” only in the differentiated progeny that are the target of the genetic disease, e.g. lymphocytes in SCID, granulocytes in CGD and monocytes/macrophages in GLD.
One way to achieve this is the use of lineage-specific transcriptional control elements, e.g. the endogenous promoter of the locus, to drive expression of the therapeutic gene in the vector. However, promoters are often spread over a long range of DNA and poorly characterized, and can thus not be easily reconstituted in their entirety in a vector construct. Furthermore, expression levels from tissue-specific promoters reconstituted in gene-transfer vectors are often not sufficient to achieve phenotypic correction, most likely because of imperfect reconstitution and/or detrimental influence of the chromatin at the semi-random vector integration site. Thus, additional strategies to regulate a transgene are direly needed.